End-stage organ failure or tissue loss is one of the most devastating and costly problems in medicine. Eight million surgeries are estimated to be performed each year incurring a health care cost of more than $400 billion annually. Organ and tissue replacement is limited by donor availability and immuno-rejection. While whole organ replacement is many years from becoming a reality, tissue replacement is currently being used for skin grafting and heart valve replacement. Current models for engineered muscle are fabricated on synthetic anchors and are limited by how well the muscle cells in culture interface with the synthetic material used. In general, as the engineered muscle construct matures and develops more passive force, the myotubes detach from the synthetic anchor and contract into a ball of cells which is useless for studying muscle function in vitro or transplantation in vivo. The creation of an engineered musculoskeletal tissue with a functional myotendinous junction (MTJ) will expand the usefulness of engineered muscle as a model for developmental muscle biology, muscle pharmacology, and for transplantation of diseased or damaged muscle. Our working hypothesis is that the introduction of functional MTJ in conjunction with applied electrical activity and mechanical strain will lead to a more advanced phenotype in the muscle and tendon; increased force production in the engineered muscles; and stronger and more developed tissue interfaces. Our purpose in this study will be to design, fabricate, and evaluate the structural and contractile characteristics of three-dimensional (3D) engineered tissues containing MTJ, one of the principal tissue interfaces required for a functional musculoskeletal construct. We will build skeletal muscle constructs co- cultured with engineered tendon constructs (ETC) or segments of adult (ART) or fetal rat tail (FRT) tendon. We hypothesize that the co-culture of tendon tissue and muscle will produce constructs with viable muscle- tendon interfaces that remain intact during force production and that will advance the myocyte differentiation within the construct. We also propose to use bioreactors to place the tendon-muscle constructs into physical environments which simulate the stress, strain, and contractile activity of hind limb muscles resembling those found in vivo. [unreadable] [unreadable] [unreadable]